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Abstract

In plants, sesquiterpenes of different structural types are biosynthesized from the isoprenoid intermediate farnesyl diphosphate. The initial reaction of the biosynthesis is catalyzed by sesquiterpene cyclases (synthases), In Artemisia annua L. (annual wormwood), a number of such sesquiterpene cyclases are active, We have isolated a cDNA clone encoding one of these, amorpha-4,11-diene synthase, a putative key enzyme of artemisinin biosynthesis. This clone contains a 1641-bp open reading frame coding for 546 amino acids (63.9 kDa), a 12-bp 5'-untranslated end, and a 427-bp 3'-untranslated sequence. The deduced amino acid sequence is 32 to 51% identical with the sequence of other known sesquiterpene cyclases from angiosperms. When expressed in Escherichia coli, the recombinant enzyme catalyzed the formation of both olefinic (97.5%) and oxygenated (2.5%) sesquiterpenes from farnesyl diphosphate. GC-MS analysis identified the olefins as (E)-beta-farnesene (0.8%), amorpha-4,11-diene (91.2%), amorpha-4,7(11)-diene (3.7%), gamma-humulene (1.0%), beta-sesquiphellandrene (0.5%), and an unknown olefin (0.2%) and the oxygenated sesquiterpenes as amorpha-4-en-11-ol (0.2%) (tentatively), amorpha-4-en-7-ol (2.1%), and alpha-bisabolol (0.3%) (tentatively). Using geranyl diphosphate as substrate, amorpha-4,11-diene synthase did not produce any monoterpenes. The recombinant enzyme has a broad pH optimum between 7.5 and 9.0 and the K-m values for farnesyl diphosphate, Mg2+, and Mn2+ are 0.9, 70, and 13 mu M, respectively, at pH 7.5. A putative reaction mechanism for amorpha-4,11-diene synthase is suggested. (C) 2000 Academic Press.

Keywords

amorpha-4,11-diene synthase; cDNA cloning; GC-MS; bacterial expression; sesquiterpenes monoterpenes; Artemisia annua

Published in

Archives of Biochemistry and Biophysics
2000, volume: 381, number: 2, pages: 173-180

SLU Authors

  • Bengtsson, Marie

    • Department of Plant Protection Sciences, Swedish University of Agricultural Sciences

UKÄ Subject classification

Biochemistry
Molecular Biology

Publication identifier

  • DOI: https://doi.org/10.1006/abbi.2000.1962

Permanent link to this page (URI)

https://res.slu.se/id/publ/110570